1. Field of the Invention
The present invention relates generally to methods of producing electrodes for the accumulation detection of thiol-binding analytes. More specifically, the present invention describes the production of analyte-accumulating electrodes having dithiol groups attached thereto, and methods for using such electrodes to accumulate and detect thiol-binding analytes in target samples.
2. Description of the Related Art
Methods and apparatus for the efficient and accurate detection and quantification of thiol-binding analyte levels in target samples are of particular interest for use in a wide range of applications. For example, the effective and efficient detection of heme or hemoglobin in human feces, i.e. fecal occult blood (FOB) detection, is of significant interest in the diagnosis of colorectal cancer. Colorectal cancer has an annual worldwide incidence of more than 600,000 cases and is the third most common human cancer. It has been reported as being the second leading cause of death in North America (Lieberman, et al. xe2x80x9cUse of Colonoscopy to screen Asymptomatic Adults for Colorectal Cancer,xe2x80x9d New England Journal of Medicine, 343, 162-168 (2000)). Among those over 45 years of age, 10% have colorectal polyps of which 1% will become malignant. Early detection of these lesions increases patient survival rates. Id. The presence of heme or hemoglobin in the feces is an indication of bleeding colon polyps which are a known risk factor for the developments of colon cancer. By monitoring the levels of heme of human feces, the early detection and treatment of colorectal cancer is more readily achieved.
Other applications for the accumulation and detection of heme include the diagnosis of malarial infection. Malaria infections can result in the accumulation of heme in infected red blood cells. By monitoring the accumulation of heme in red blood cells, the early detection of malarial infections can be achieved.
Several methods for the detection of heme in a sample are available commercially and used clinically. For example, fecal occult blood detection methods are available under the tradenames Hemoccult II and Hemoccult II SENSA from Smith Kline Diagnostic, Palo Alto, Calif., and immunochemical detection methods are available under the tradenames Hemeselect and FlexSure OBT. Unfortunately, such methods tend to lack the desired sensitivity and specificity to avoid high false positive detection rates for fecal occult blood.
Other methods for accumulating thiol-binding analytes such as iron protoporphyrin and iron hematoporphyrin using dimercaptoalkane-modified solid wire or plate gold electrodes have been disclosed in xe2x80x9cElectrochemistry of Self-Assembled Monolayers of Iron Protoporphyrin IX Attached to Modified Gold Electrodes through Thioether Linkagexe2x80x9d D. L. Pilloud, et al., J. Phys. Chem. B 2000, 104, 2868-2877, incorporated herein by reference. However, as discussed by Pilloud, the electrodes produce for use therein are disadvantageous in that the thiolated electrode surfaces tend to degrade relatively rapidly when the electrodes are left in contact with air or immersed in aqueous solution. Id. at 2869. Accordingly, such methods are unsuitable for producing electrodes capable of accumulating analytes for relatively long periods of time (for example one or more days) and capable of being transported in air or water for any significant period of time.
The present invention overcomes the aforementioned disadvantages by providing methods of producing electrodes comprising stable thiolated surfaces, and methods of using such electrodes to accumulate and detect thiol-binding analytes, especially heme, in a target sample with high degree of sensitivity and selectivity. In particular, applicants have discovered that the thiolated surfaces of the electrodes produced via the present inventions tend to be advantageously stable, i.e. avoid significant degradation, for periods of time as long as several hours to one or more days (or longer) either in the presence or absence of oxygen. Although applicants do not wish to be bound by or to any particular theory of operation, it is believed that the present methods provide electrodes which overcome the relative instability of prior art electrodes in the presence of oxygen by preparing the electrode surface through an electrochemical treatment prior to thiolating the surface. Tests were conducted which comprised aerating a target sample solution comprising heme and introducing an electrode of the present invention thereto. The tests showed that heme was as easily attached to the electrode in such solution as it is in specially de-aerated solutions, suggesting that the bonds formed between dithiol molecules and the electrode substrate according to the present methods do not readily break in the presence of oxygen.
Because of the aforementioned surface stability, the electrodes produced herein can be used advantageously according to the present invention to accumulate and detect amounts of thiol-binding analytes from low concentration analyte solutions with greater accuracy than prior art electrode processes. To ensure sufficient interaction of thiol-binding analyte molecules in relatively low concentration analyte solutions (for example, those having a concentration measured in nanomolar (nM) or even smaller units) with an electrode for the concentration and accurate detection thereof, it is often necessary to allow the electrode to remain in the target analyte solution for a period of time as long as several hours to one or more days. While many prior art electrodes tend to degrade before such necessary interaction times are achieved, the electrodes produced herein tend to be sufficiently stable to remain in solution for periods of time necessary to measure low analyte concentrations with an accuracy not previously achievable using prior art methods. Applicants have recognized, for example, that the electrode of the present invention can be used to detect thiol-binding analytes in solutions comprising an analyte concentration of greater or less than about 100 micromolar (xcexcM). In certain embodiments, the present methods can be used to detect analytes in solutions as low as from about 10 nM to about 100 xcexcM of analytes. Preferably, the present methods are capable of detecting analytes in solution comprising concentrations as low as less than about 10 nM analytes, and even more preferably less than about 1 nM analytes.
Applicants have further recognized that the electrodes having analyte accumulated thereon produced according to the present methods tend to be sufficiently stable to allow the electrode to be transferred from a sample solution to a test solution for use in analyte detection. By concentrating analyte samples onto an electrode and/or transferring the analyte into another solution, the present methods allow for a more sensitive, selective, and accurate detection of low analyte concentrations in sample solutions than is obtainable using prior art electrode methods. In addition, the accumulated-analyte electrodes can be transported in air or aqueous solution from, for example, a field testing site to the laboratory for analysis. This obviates the need to transport entire liquid samples, such as blood samples, which may require refrigeration or other handling and transport considerations, for testing to the laboratory.
According to certain embodiments of the present methods, applicants have also recognized that the production and use of electrodes having a fractal dimension (Df) of greater than about 2 allows for the detection of analytes in solution with greater sensitivity than prior art methods. As will be recognized by those of skill in the art, the term xe2x80x9cfractal dimensionxe2x80x9d refers to a measurement of fractal geometric dimension. For example, a metal electrode with a flat surface has a Df=2. As discussed below, certain metal electrodes comprising coiled metal wires (in some cases with surfaces roughened via cyclic voltammetry) produced via the present methods have Df values of greater than 2. By using electrodes having Df greater than 2, certain preferred embodiments of the present invention allow for the binding of greater amounts of dithiol compounds, and thus, greater amounts of analyte, to the electrode for the detection of analyte with greater accuracy and sensitivity than prior art methods.
According to one aspect, the present invention provides methods of producing an electrode comprising: providing a substrate capable of binding a dithiol molecule thereto; electrochemically treating the substrate to provide a treated substrate having a fractal dimension of greater than about 2; and contacting the treated substrate with dithiol molecules to produce an electrode having dithiol groups attached thereto and capable of binding an analyte thereto.
According to another aspect, the present invention comprises methods of accumulating an analyte capable of bonding to a dithiol moiety onto an electrode comprising: providing an electrode of the present invention capable of binding the analyte to be detected thereto; and contacting the electrode with a target solution comprising an analyte to bind at least a portion of the analyte to the electrode.
According to yet another aspect, the present invention provides methods of detecting analytes in a target solution comprising: providing an electrode of the present invention capable of binding the analyte to be detected thereto; contacting the electrode with a target solution comprising an analyte to bind at least a portion of the analyte to the electrode; and detecting the analyte on the electrode.